BLOOD SUBSTITUTES
Transfusion of a patient with donated blood has a number of disadvantages. Firstly, there may be a shortage of a patient's blood type. Secondly, there is a danger that the donated blood may be contaminated with infectious agents such as hepatitis viruses, cytomegalovirus, Epstein-Barr virus, serum parvoviruses, syphilis, malaria, filariasis, trypanosomiasis, babsiosis, pathogenic bacteria, and HIV (Bove, 1986, Progr. Hematol. 14:123-145). Thirdly, donated blood has a limited shelf life.
An alternative to transfusion involves the use of a blood substitute. A blood substitute is an oxygen carrying solution that also provides the oncotic pressure necessary to maintain blood volume. Two types of substitutes have recently been studied, fluorocarbon emulsions and hemoglobin solutions.
Fluorocarbons however are not feasible blood substitutes, since they are known at times to block the natural immune system (Dellacherie, 1986, Crit. Rev. Ther. Drug Carriers 3:41-94). In addition, the use of fluorocarbons is limited to situations in which high partial pressures of oxygen can be administered. They do not have a sufficiently high oxygen binding capacity for use under normal physiological conditions.
Hemoglobin as it exists within the red blood cell is composed of two alpha-like globin chains and two beta-like globin chains, each with a heme residue. One alpha-like globin chain and one beta-like globin chain combine to form a dimer which is very stable. Alpha-like and beta-like globin genes are each a family of related globin genes which are expressed at different stages of development and regulated by oxygen tension, pH, and the development from embryo to fetus to newborn. Two dimers then line up in antiparallel fashion to form tetramers. The binding of dimers to form the tetramers is not as strong as in the case of monomers binding to associate into dimers. The tetramers, therefore, have a tendency to fall apart to form dimers and there is always an equilibrium between tetramers, dimers, and monomers. At high concentrations of globin, the predominant form is the tetramer; with dilution, the dimer becomes the predominant form. This equilibrium is also affected by solvent, salts, pH and other factors as the forces binding the monomers together are primarily electrostatic.
Hemoglobin may exist under two conformations, the oxygenated (R-form) or deoxygenated (T-form). The deoxy structure is stabilized by the formation of salt bridges involving definite amino and carboxylic groups of globins.
The oxygen binding characteristics of hemoglobin can be characterized by a curve, called the oxygen affinity curve, obtained by plotting the fractions of available hemoglobin sites saturated with oxygen as a function of the partial pressure of oxygen in equilibrium with the solution. Information may be obtained from such plots regarding the cooperativity of oxygen binding to hemoglobin using the following Hill equation: EQU Y/1-Y=K[O.sub.2 ].sup.n
where Y is the fraction of sites occupied by oxygen and n and K are adjustable constants. When the cooperativity of the oxygen binding to hemoglobin is modified, this results in a decrease of the Hill Coefficient (n). Therefore, the value of the Hill coefficient can be considered as a useful reflection of the efficacy of the oxygen-carrying function. The oxygen affinity of hemoglobin may also be characterized by determining the P.sub.50, which is the partial oxygen pressure which leads to 50% saturation.
The alpha-like globin genes of hemoglobin are clustered together on chromosome 16 and include genes encoding the embryonic zeta globin chain and the adult alpha globin chain, present in both the fetus and newborn. The beta-like globin genes reside on chromosome 11 and include genes encoding the embryonic epsilon-globin chain, the fetal gamma-globin chain, and the adult delta-globin and adult beta-globin chains. Two types of gamma globin chains have been identified, .sup.G gamma and .sup.A gamma, which differ by the presence of a single glycine of alanine residue, respectively, at amino acid 135 (Scroeder et al., 1968, Proc. Natl. Acad. Sci. U.S.A 60: 537-544). The gamma chain has been found to contain a polymorphic site at position 75, which also can be occupied either by isoleucine or threonine. A variety of hemoglobins may be formed (reviewed in Kutlar et al., 1989, Hemoglobin 13:671-683 and Honig and Adams, Human Hemoglobin Genetics, Springer Verlag, N.Y. pp, 29-33). Examples of hemoglobins (Hb) include hemoglobin A (HbA-alpha.sub.2 beta.sub.2), HbA.sub.2 (alpha.sub.2 delta.sub.2), HbF (alpha.sub.2 gamma.sub.2 ), Hb Barts (gamma.sub.4), HbH (beta.sub.4), and Hb Portland I (zeta.sub.2 gamma.sub.2), Hb Portland II (zeta.sub.2 beta.sub.2), Hb Portland III (zeta.sub.2 delta.sub.2) Hb Gower I (zeta.sub.2 epsilon.sub.2), and Hb Gower II (alpha.sub.2 epsilon.sub.2).
There are obstacles however to using native hemoglobin as a blood substitute. Firstly, large dosages are required (Walder, 1988, Biotech '88, San Francisco, Nov. 14-16, 1988). A single unit (450 ml) of a 10% hemoglobin solution contains 45 g of protein. It is estimated that ten million units of blood are used in the U.S. per year. Therefore, the production of 450,000 kg of hemoglobin per year would be required. Secondly, it is important to obtain hemoglobin that is free from infectious agents and toxic substances. Thirdly, although hemoglobin is normally a tetramer of 64,000 molecular weight, it can dissociate to form alpha-beta dimers. The dimers are rapidly cleared by the kidneys and the residence time is much too short for cell-free hemoglobin to be useful as a blood substitute. Fourthly, cell-free hemoglobin has too high an oxygen affinity to effectively release oxygen to the tissue due to the absence of 2,3-diphosphoglycerate (2,3DPG). Efforts to restore 2,3-DPG have been unsuccessful since 2,3-DPG is rapidly eliminated from the circulation.